During excitation-contraction (EC) coupling a cardiac myocytes in influx of Ca2+ ions carried by the Ca2+ current (lca) activates a large release of Ca2+ from the sarcoplasmic reticulum (SR) vi the SR Ca2+ release channel (or ryanodine receptor, RYR). The subsequent declining phase of this {Ca2+}, transient is due in part, to SR Ca2+ reuptake mediated by the SR Ca2+ ATPase (SERCA2A). The Principal Investigators have discovered only recently that calcineurin (CaN), a calmodulin-activated enzyme, acting alon and/or in concert with FK506 binding proteins (FKBPs), regulates the {Ca2+}, transient and the frequency of elementary SR Ca2+ release events ("Ca sparks"). Calcineurin does this in the absence of changes in L-type Ca2+ current. The planned work will study this important new modulator at the cellular and molecular level by state-of-the-art techniques. The project will focus on the following inter-related specific aims that provide links between the biophysics and the underlying molecular cell biology. (1) How does CaN altar Ca2+ signaling in rat ventricular heart cells? New results will be extended with patch clamped myocytes to determine how elevation or inhibition of CaN activity alter whole cell currents, SR Ca2+ load, the [Ca2+], transient, and contractility. Effects inhibitors will be examined in biochemical assays. (2) How are Ca2+ sparks, the elementary SR Ca-release events, altered by CaN? Confocal microscopy will be used to study effects of CaN on the frequency and kinetics of Ca2+ sparks to provide a precise complement to the results of (1) above. (3) What is the impact of SR calcium load on the action of CaN? SR load will be varied by use of phospholamban-difficient transgenic mice or by transient overexpression of the SERCA2a by the use of the Pl's new novel adenovirus component transfection system. Effects of CaN will be examined as in (1) and (2) above. (4) How does FKBP effect the action of CaN and SR Ca release? The results of (1), (2) and (3) will be used to compare the action of FKBP-activating ligands in parallel studies with normal and CaN-inhibited cells. In summary, the plan is an integrated strategy of biochemistry, gene transfection, single cell voltage clamp, and high resolution confocal Ca2+ imaging that will define novel SR regulatory mechanisms in heart. This project will provide an understanding of the changes in SR function that underlie the contractile dysfunction of diseases including cardiomyopathies and hypertrophy.